Compositions of drug delivery agents and methods of use thereof

This application claims priority from U.S. Provisional Application Ser. No. 62/649,735 filed on 29 Mar. 2018, which is incorporated herein by reference in its entirety.

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The present disclosure generally relates to methods and compositions for drug delivery in antibacterial and anticancer therapies.

The Center for Disease Control and Prevention (CDC) estimates that each year over 2,000,000 reported cases of illnesses and 23,000 deaths are caused by antibiotic resistance in the United States (US). The World Health Organization (WHO) has identified antibiotic resistance as being one of the biggest threats to global health and food security. This urgent threat of multi-drug resistant (MDR) bacteria, combined with the fact that pharmaceutical companies have downsized their R&D efforts to pursue new antibiotics, has resulted in a broad-sweeping panic caused by the fear of a worldwide epidemic, a scenario which prompted the former Director-General of the WHO—Dr. Margaret Chan—to state that, “The world is heading towards a post-antibiotic era in which common infections will once again kill. [ . . . ] This may even bring the end of modern medicine as we know it.”

The traditional approach to solve this growing problem typically relies on developing structural analogs of existing antibiotics, pursuing new leads, or perhaps revisiting previous compounds that were at one point considered extremely toxic. Combination therapy that combines multiple antibiotics into one treatment is also a common strategy to overcome resistance in bacteria; however, it can also be quite toxic. In recent years, these approaches have not only been outpaced by the onset of resistance, but the situation is exacerbated by the fact that the number one reason most antibiotics fail is due to a lack of solubility in water and their inability to permeate bacterial cell membranes.

Furthermore, treatments for aggressive and metastatic types of cancer often come in the form of “cocktails” that consist of multiple small-molecule anti-cancer drugs that are often administered multiple times intravenously over the course of a lengthy treatment regimen. One example of this combination-based strategy is FOLFIRINOX—consisting of Folinic acid, Fluorouracil, Irinotecan, and Oxaliplatin—which is often used to treat metastatic pancreatic cancer. Although it is an effective treatment capable of extending the median overall survival rate to 11.1 months, compared to only a few months with other single-drug-based systems, FOLFIRINOX is also incredibly toxic and may result in an increased rate of infection due to a drop in white blood cells, tiredness as a result of a lower red blood cell count, the formation of ulcers, hair loss, and so on. The drug delivery community has worked to develop systems capable of delivering anti-cancer drugs with improved pharmacokinetics and dramatically reduced toxicities; however, it remains a major challenge to construct platforms that can support the precise loading of three or more drugs without unintentional early release of drugs. Even more troubling, there are also now concerns emerging over how drugs that are conjugated directly to the delivery platform are not released completely and efficiently once inside the tumor, and even when they are released in vitro/vivo, their mode of action may be different than what is expected in comparison to the observed mechanisms of small-molecule free drugs. These major delivery issues can often perturb or completely derail clinical trials that investigate efficacy using modern drug delivery systems.

Among the various aspects of the present disclosure is the provision of compositions and methods related to drug-loaded nanoparticles comprising polymers (e.g., comprising various norbornene (Nb)-based monomers) used for the treatment of bacterial infections and or cancer.

An aspect of the present disclosure provides for a composition comprising a polymer-based drug-delivery agent, wherein the polymer-based drug-delivery agent comprises at least a first polymerized monomer and at least a second polymerized monomer; the first polymerized monomer comprises at least a first macrocyclic moiety and the second polymerized monomer comprises a second macrocyclic moiety; the first macrocyclic moiety is non-covalently bound or is capable of non-covalently binding a first antibiotic or a first anticancer agent; and/or the second macrocyclic moiety is non-covalently bound or is capable of non-covalently binding a second antibiotic, a second anticancer agent, an alkali metal cation, or a transition metal cation.

In some embodiments, a first macrocyclic moiety is non-covalently bound to a first antibiotic and the second macrocyclic moiety is non-covalently bound to a second antibiotic and the first antibiotic and the second antibiotic are not the same antibiotic.

In some embodiments, a first macrocyclic moiety is non-covalently bound to a first antibiotic and the second macrocyclic moiety is non-covalently bound to a an alkali metal cation or a transition metal cation.

In some embodiments, a first macrocyclic moiety is non-covalently bound to a first anticancer agent and the second macrocyclic moiety is non-covalently bound to a second antibiotic and the first anticancer agent and the second anticancer agent are not the same anticancer agent.

In some embodiments, a polymer-based drug-delivery agent comprises: a polymerized monomer comprising a protonatable subunit; a polymerized monomer comprising an imaging agent (NIR dye, contrast agent), a targeting group (e.g., folate to target breast cancer cells, peptide), or a binding group; a masked polymerization initiator, capable of being deprotected and initiating a polymerization; a glucosamine derivative; a norbornene group (e.g., a N-alkyl-5-exo-norbornene-2,3-dicarboxylic acid imide group); or a norbornene (Nb)-based hexaethylene glycol capped with an N-L-arginine-glucosamine subunit (Nb-LARGE monomer, Nb-Arg).

In some embodiments, the polymer-based drug-delivery agent is capable of self-assembly into a micelle.

In some embodiments, the composition comprises a non-covalent cross-linking group (e.g., adamantane-functionalized crosslinker, bis(adamantyl) crosslinker) capable of non-covalently binding two or more macrocyclic moieties.

In some embodiments, the first macrocyclic moiety is a cyclodextrin bound to an antibiotic and the second macrocyclic moiety is a calixarene bound to an Ag.

In some embodiments, the first macrocyclic moiety is a cyclodextrin bound to a first anticancer agent and the second macrocyclic moiety is a cyclodextrin bound to a second anticancer agent, wherein the first anticancer agent is not the same as the second anticancer agent.

In some embodiments, the polymer-based drug-delivery agent is easily protonated, targets a bacteria cell surface, or destabilizes cell membranes; is capable of self-assembly; exhibits a controlled and sustained delivery of a silver ion (Ag) and one or more small-molecule antibiotic simultaneously; comprising Aginduces oxidative stress in bacteria; comprising Agdisrupts disulfide bond formation in proteins, resulting in an increase in permeability of their cellular membranes; or induces collagen synthesis during wound healing.

In some embodiments, the polymer-based drug-delivery agent comprises a polynorbornene backbone.

In some embodiments, the macrocyclic moiety comprises a hydrophobic cavity capable of binding a small molecule drug or alkali metal cations or transition metal cations with high affinity (e.g., for antibiotics or anticancer drugs, a Kof greater than about 100 Mand for alkali metal cations or transition metal cations, a Kof greater than about 10M).

In some embodiments, the first macrocyclic moiety or second macrocyclic moiety is independently selected from the group consisting of: a cyclodextrin; a β-cyclodextrin; a calixarene; a thiacalix[n]arene; a tert-butyl-thiacalix[4]arene; a cyclophane; a crown ether; and a [18]-crown-6 ether.

In some embodiments, the first anticancer agent and the second anticancer agent are independently selected from one or more of the group consisting of: camptothecin; doxorubicin; cisplatin; oxaliplatin; 5-fluoruracil; chlorambucil; methotrexate; and irinotecan HCl.

In some embodiments, the first antibiotic or the second antibiotic are independently selected from one or more of the group consisting of: a β-lactam; amoxicillin; imipenem; an aminoglycoside; a quinolone; a fluoroquinolone; Levofloxacin; chloramphenicol; a sulfonamide; Sulfadiazine, Sulfamethoxazole; tetracycline; linezolid; and a thiol NDM-1 inhibitor.

In some embodiments, the transition metal cation is selected from a copper (Cu) ion or a silver (Ag) ion.

In some embodiments, the polymer-based drug-delivery agent comprises: a PEG (e.g., a Nb-PEG-X monomer); a hexaethylene glycol capped with an N-L-arginine-glucosamine subunit (e.g., a Nb-LARGE monomer, Nb-Arg); a methyl (e.g., an Nb-Me monomer); or a norbornene (Nb)-based monomer.

In some embodiments, the first macrocyclic moiety or the second macrocyclic moiety is a calixarene comprising a thioether-pyridine bridge, wherein the thioether-pyridine bridge increases binding of the transition metal cation inside the calixarene compared to a calixarene without the thioether-pyridine bridge.

Another aspect of the present disclosure provides for a method of producing a polymer-based drug-delivery agent described herein, comprising: generating at least two monomers comprising a backbone and a first macrocyclic moiety and a second macrocyclic moiety; and polymerizing the monomers, wherein the first macrocyclic moiety and the second macrocyclic moiety are non-covalently bound to different therapeutic agents.

In some embodiments, the method comprises linking a targeting agent or imaging agent to the backbone.

In some embodiments, a polymerization is performed using a ratiometric ring-opening metathesis polymerization (ROMP) reaction; the ROMP reaction is conducted using Grubbs' third-generation ruthenium catalyst in non-polar tetrahydrofuran (THF) solvent; or a monomer has an active or ‘living’ ruthenium species on one end.

In some embodiments, the method comprises generating an amino-functionalized cyclodextrin comprising mono-tosylating a cyclodextrin, resulting in a cyclodextrin with at least one tosyl group, nucleophilic displacement of the at least one tosyl group with ethylene diamine, resulting in an amino-functionalized cyclodextrin, wherein the amino-functionalized cyclodextrin is reacted with a norbornene-based acyl chloride, resulting in a norbornene-functionalized cyclodextrin.

In some embodiments, the method comprises generating a norborene-functionalized calixarene by generating a 1,2 calixarene conformer via a Mitsunobu reaction between a calixarene and a ortho-substituted norbornyl-pyridine with methylenethio ethanol linkers and functionalizing remaining tert-butyl phenol subunits of the calixarene ring with triethylene glycol monomethyl ether chains, resulting in improved solubility of a monomer in tetrahydrofuran (THF).

In some embodiments, the method comprises generating a tert-butyloxycarbonyl (Boc)-protected arginine monomer (Nb-Arg (Boc)) to serve as a precursor to a positively charged block of a functional copolymer.

In some embodiments, the method comprises generating a macromonomer consisting of poly(ethylene glycol) (PEG3000) bearing a norbornene group at one end (Nb-PEG).

In some embodiments, the method comprises initiating polymerization of a monomer comprising deprotecting a masked polymerization resulting in a three-dimensional polymer network.

Yet another aspect of the present disclosure provides for a method for treating a subject in need thereof comprising administration of an effective amount of a polymer-based drug-delivery agent to a patient in need thereof, wherein the polymer-based drug-delivery agent is loaded with a therapeutic agent and the subject has cancer, a bacterial infection, or a wound.

In some embodiments, subject has cancer, the cancer is selected from one or more of the group consisting of: pancreatic cancer, breast cancer, leukemia, lymphoma, ovarian cancer, and metastatic cancer;

In some embodiments, subject has a bacterial infection, the bacterial infection selected from or more of the group consisting of: gram-positive bacteria, gram-negative bacteria, multi-drug resistant-bacteria (MDR), methicillin-resistant(MRSA),, or NDM-1 producing carbapenem-resistant Enterobacteriaceae.

In some embodiments, subject has a wound.

In some embodiments, cancer cells are targeted with a targeting agent; folate to target breast cancer cells; or peptides.

In some embodiments, anti-cancer drugs are released after administering the polymer-based drug-delivery agent to the subject.

Yet another aspect of the present disclosure provides for a device (e.g., a dual-syringe) used to treat hemorrhages and prevent bacterial infections of open wounds, wherein a first syringe delivers sodium alginate, which is a rapid hemostat; and/or a second syringe delivers the composition according to claim(e.g., drug-loaded nanoparticles).

Other objects and features will be in part apparent and in part pointed out hereinafter.

The present disclosure is based, at least in part, on the discovery that a polymer-based drug-delivery agent (e.g., nanoparticles constructed from norbornene (Nb)-based monomers) can be used for the delivery of drugs (or therapeutic agents) and small molecules to, for example, treat multidrug resistant bacteria or aggressive forms of cancer. Another application can be for agriculture seed coatings for protection and seedling growth.

Described herein is a next-generation universal plug-and-play drug delivery system comprised of linked supramolecular monomers that can act as ‘smart’ receptors for a wide variety of small-molecule therapeutics, such as anti-cancer or anti-bacterial drugs that can be polymerized together in precise monomer ratios to yield combination drug-loaded nanoparticles. This ‘supramacromolecular’ approach can completely bypass any issues associated with prodrug-to-drug conversion in vitro/vivo because the unmodified drug itself will be what is loaded into the receptors that are positioned along the polymer chain, and therefore the drug's mechanism of action will remain undistorted. Additionally, the small-molecule drugs will not need an external event in vitro/vivo to trigger their release from the platform, but rather the release will be governed by the non-covalent binding interactions between the receptor and the drug.

The difference between previous studies and the presently disclosed invention relates specifically to the method or manner in which the drugs and metal ions are incorporated into the polymer-based platform. Previous studies employed a method of physically (covalently) attaching the drugs to the monomer prior to polymerization and then relied on natural enzymes in an organism to cleave the covalent bonds and release the drugs. The presently disclosed approach does not chemically modify the drugs, but instead loads the free drug form into receptors that bind the drug or metal ion temporarily and slowly releases them over time. This distinction is significant because many drug-conjugated monomers and polymers often fail to release their cargo in vivo, and for some of the ones that do, they sometimes lose their original mechanism of action in the site of release (e.g., the site of infection).

Here is described the synthesis of supramolecular monomers and polymers. Also demonstrated is that supramolecular monomers and polymers can bind a wide variety of antibacterial and anticancer drugs and a metal ion e.g., silver (Ag)). Described herein is in vitro efficacy data showing that the drug-loaded polymer platform can kill bacteria and inhibit further growth over time (see e.g., Example 4).

The present disclosure provides for a universal platform for the delivery of any combination of small-molecule drugs. Usually small-molecule drugs are not water-soluble and/or are toxic, the latter of which is especially true when used in drug cocktails consisting of multiple drugs, which is the current practice employed in the clinic for both multidrug-resistant (MDR) bacterial infections and aggressive forms of cancer. The significance of the presently disclosed system is that it is not necessary to remake a new polymer to deliver different combinations of drugs. Here, one would simply choose the desired drug combination and precise ratio and add it to the disclosed polymers. The resultant drug-loaded polymers could then be used as a broad-spectrum antimicrobial that could be used as a broad-spectrum treatment against MDR bacterial infections or as a non-toxic chemotherapy for treatment of aggressive forms of cancer. Furthermore, the selective capture and slow release of small-molecule drugs or nutrients are ideal for agricultural applications, such as seed coatings for protection and seedling growth.

Overcoming Antibiotic Resistance

The present disclosure provides for compositions and methods for overcoming antibiotic resistance. The Center for Disease Control and Prevention (CDC) estimates that each year over 2,000,000 reported cases of illnesses and 23,000 deaths are caused by antibiotic resistance in the United States (US).

The continual rise of antibiotic resistance—coupled with the shrinking pipeline of new antibiotics—poses a major threat to global health. The traditional approach to solve this growing problem typically relies on developing structural analogs of existing antibiotics, pursuing new leads, or even revisiting previous compounds that were at one point considered very toxic. Combination therapy that combines multiple small-molecule antibiotics into one treatment is also a common strategy to overcome resistance in bacteria, however, it can also be quite toxic. In recent years, these approaches have not only been outpaced by the onset of resistance, but the situation is exacerbated by the fact that the number one reason most antibiotics fail is due to a lack of solubility in water and their inability to permeate bacterial cell membranes. Here is described a different strategy; one which involves precise supramolecular receptors built into a polymer platform, where each receptor can be loaded with different combinations of antimicrobial agents, depending on the type of multidrug-resistant bacteria that is to be treated. Moreover, this platform is easily converted into larger nano-based structures, which stabilize the loaded drugs and allows for selective targeting of bacteria through specific cell surface-based interactions. Since no pro-drugs are involved, the mechanism of action of each agent is unperturbed and each undergoes slow release in a predictable manner. Lastly, described herein, are in vivo efficacy studies against different drug-resistant bacterial strains, and how this next-generation platform is an ideal material for sustained protection that may prove useful in wound healing applications, among others.